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Direct Pathway Cloning of Neglected Bacteria in the Hunt for Novel (Bio-)Chemistry

Fact Sheet

Result in Brief

Results

Project information

DiPaC_MC

Grant agreement ID: 745435

Start date

1 September 2017

End date

31 August 2019

Funded under:

H2020-EU.1.3.2.

Overall budget:

€ 159 460,80

EU contribution

€ 159 460,80

Coordinated by:

TECHNISCHE UNIVERSITAET MUENCHEN

Germany

Objective

To overcome recent global health threats, such as antibiotic resistance, a resurgence in the discovery of new chemical and thus biomedical diversity from microbial sources is needed. Genome mining in combination with heterologous expression is an approach that will overcome these challenges. It involves incorporation of yet uncharacterized natural product genetic loci into a fast growing heterologous host. For this approach, a prolific source of novel natural product gene clusters is vital. Our collaboration with the Zentralinstitut für Ernährungs- und Lebensmittelforschung (ZIEL, Technische Universität München) provides direct access to bacterial isolates completely unexplored in regards to their potential for natural product biosynthesis. Our genome analyses have revealed that many of the ZIEL isolates harbor a large number of uncharacterized natural product gene clusters. Thus, the aim of this proposal is to heterologously express natural product gene clusters from these neglected bacteria to discover novel natural product (bio-)chemistry. Because these organisms are poorly studied, the likelihood of discovering rare or novel biochemistry is immensely increased. Here, we will utilise a novel combination of synthetic biology techniques referred to as Direct Pathway Cloning. This will enable expression vectors to be constructed by large-amplicon PCR (up to 20kb) coupled to Gibson assembly. Development of the methodology is set to revolutionise synthetic biology and metabolic engineering. Downstream outcomes of this proposal will be the identification of novel, potentially bioactive natural products, the characterisation of unusual biochemistry and the addition of enzymes to the ‘biocatalytic toolkit’ for natural product synthesis and structural alteration.

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The rising incidence of antimicrobial drug resistance has spurred research on the identification of novel natural products. European scientists have developed a methodology to fast track the identification of novel natural products of cryptic gene clusters from previously understudied microorganisms.

Microbial-based natural products or metabolites have long served as the basis for many drugs including antibiotics. However, they are losing activity due to the emergence of resistance among pathogenic bacteria.

A novel approach for cloning microbial biosynthetic gene clusters

Traditionally, the discovery of novel natural molecules involves the cultivation of bacteria from large strain collections and the chemical analysis of the produced metabolites, a very tedious, expensive and time-consuming procedure. Undertaken with the support of the Marie Skłodowska-Curie programme, the DiPaC_MC project used genome sequencing in combination with new synthetic biology technologies to search for novel molecules.
“We know that microorganisms have the genetic capability to produce many more natural products than have been identified, usually because the genes may be turned off in the laboratory setting,″ explains the project fellow Paul D'Agostino. To overcome this limitation, he exploited microorganisms that had traditionally been neglected in terms of natural product biosynthesis but were likely to genetically encode large numbers of natural product biosynthetic gene clusters.
D'Agostino sequenced the genomes of these microorganisms and developed a new synthetic biology strategy to efficiently capture biosynthetic gene clusters and activate them in an alternative, well-studied bacterial host. Since many of these genetic elements remain inactive in the native organism, placing them within a second host and under control of different regulation mechanisms, he was able to artificially switch-on the biosynthetic gene clusters and identify the encoded natural products.
Known as Direct Pathway Cloning (DiPaC), this approach was able to overcome many of the limitations of previous methodologies by enhancing speed and success rates. The methodology enables the capture, restructuring and expression of biosynthetic pathways in a matter of weeks.
DiPaC_MC work generated genome sequences for a range of microorganisms. As predicted, bioinformatics analysis showed that many of these organisms encoded multiple biosynthetic gene clusters responsible for producing unique natural products. Over 200 such biosynthetic gene clusters have been identified to date.

Clinical impact of the DiPaC_MC project

“With drug resistance threatening to become a major global issue in the near future and pharmaceutical companies closing drug discovery programmes, it has been left to academia to fill the void of novel antibiotic discovery,″ emphasises D'Agostino. The DiPaC methodology offers a novel molecule discovery approach based on bioinformatic predictions of chemical novelty of the end product. Importantly, DiPaC enables the activation of these biosynthetic gene clusters more quickly than traditional methods. Moreover, by placing the genes in a faster growing host, higher product titres may be obtained.
Using well-established molecular techniques, the DiPaC methodology is easily transferable to other laboratories. With many scientific teams already requesting to use the technology, DiPaC is expected have a significant impact towards the prompt discovery of bioactive natural products.
DiPaC can also be used to determine enzyme function of particularly interesting proteins as well as to generate compound analogue libraries in the hope of increasing bioactivity. “Implementing the DiPaC methodology on phylogenetically distinct microorganisms will expedite the discovery of novel natural products and biochemistry, thereby increasing the likelihood of discovering molecules with pharmaceutical significance,″ concludes D'Agostino.